Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line

Autores
Bermúdez, Vicente; Asatryan, Asatryan A.; Mukherjee, P. K.; Giusto, Norma Maria; Bazan, Nicolás Guillermo; Mateos, Melina Valeria
Año de publicación
2021
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
The retinal pigment epithelium (RPE) plays critical roles in the correct function of the neural retina and photoreceptor survival. Classical phospholipases D (PLD1 and 2) hydrolyze phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline. PA can be further dephosphorylated to diacylglycerol (DAG) by lipid phosphate phosphatases (LPPs). DAG and PA, as bioactive lipids, can modulate the activity of various proteins involved in cell signaling events, such as protein kinases C and the mTOR (mammalian target of rapamycin) complex, among others. Our previous studies demonstrated for the first time the participation of classical PLDs in the inflammatory response and in the autophagic process of RPE (ARPE-19 and D407) cells exposed to lipopolysaccharide (LPS). The aim of the present work was to study PLD1 and PLD2 expression in a new human RPE cell line (ABC cells), spontaneously arisen from a primary RPE cell culture. Western blot assays (WB) show that both classical PLDs are expressed in ABC cells. Using PLD1 and PLD2 siRNA, we were able to partially decrease the expression of PLD1 (by 42%) and PLD2 (by30 %). Since PLD-generated PA activates mTORC1, the main inhibitor of autophagy initiation, we wanted to study the effect of classical PLDs silencing on mTOR activation. For this purpose, WB assays were performed in order to study the mTOR downstream effector S6 kinase (S6K) activation (phosphorylation) in ABC cells transfected with PLD1 and PLD2 siRNA. Our results show that in ABC cells transfected with PLD1 and PLD2 siRNA S6K activation is reduced by 34%. This result is in accordance with the increased autophagic process induced by PLD1 and PLD2 pharmacological inhibitors, previously observed in D407 RPE cells. Since it was previously demonstrated that the PLD pathway can modulate the phagocytic process in macrophages, we wanted to evaluate the effect of PLD1 and PLD2 silencing on the photoreceptor outer segment (POS) phagocytic process in ABC cells. With this aim, ABC cells were incubated with POS for 16 h, and total (bound + internal) and internalized POS were measured by WB using an anti-rhodopsin antibody. Under basal conditions, PLD1 and PLD2 silencing seem not to significantly affect POS phagocytosis by ABC cells. In conclusion, our results demonstrate the expression of classical PLD isoforms in a new RPE cell line and their role in the modulation of the mTOR/S6K pathway. Further experiments are needed to fully elucidate the role of classical PLDs in the phagocytic process of ABC RPE cells exposed to inflammatory conditions. The results presented herein, together with our previous findings, contribute to the knowledge of the molecular basis of retinal inflammatory and degenerative diseases, such as diabetic retinopathy, aged-related macular degeneration and endophthalmitis, among others.
Fil: Bermúdez, Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Asatryan, Asatryan A.. Louisiana State University Health Sciences Center New Orleans (lsu Health);
Fil: Mukherjee, P. K.. Louisiana State University Health Sciences Center New Orleans (lsu Health);
Fil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Bazan, Nicolás Guillermo. Louisiana State University Health Sciences Center New Orleans (lsu Health);
Fil: Mateos, Melina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology
Argentina
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Sociedad Argentina de Microbiología General
Materia
PHOSPHOLIPASE D
PHAGOCYTOSIS
RETINAL PIGMENT EPITHELIUM
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/229410

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spelling Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell lineBermúdez, VicenteAsatryan, Asatryan A.Mukherjee, P. K.Giusto, Norma MariaBazan, Nicolás GuillermoMateos, Melina ValeriaPHOSPHOLIPASE DPHAGOCYTOSISRETINAL PIGMENT EPITHELIUMhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The retinal pigment epithelium (RPE) plays critical roles in the correct function of the neural retina and photoreceptor survival. Classical phospholipases D (PLD1 and 2) hydrolyze phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline. PA can be further dephosphorylated to diacylglycerol (DAG) by lipid phosphate phosphatases (LPPs). DAG and PA, as bioactive lipids, can modulate the activity of various proteins involved in cell signaling events, such as protein kinases C and the mTOR (mammalian target of rapamycin) complex, among others. Our previous studies demonstrated for the first time the participation of classical PLDs in the inflammatory response and in the autophagic process of RPE (ARPE-19 and D407) cells exposed to lipopolysaccharide (LPS). The aim of the present work was to study PLD1 and PLD2 expression in a new human RPE cell line (ABC cells), spontaneously arisen from a primary RPE cell culture. Western blot assays (WB) show that both classical PLDs are expressed in ABC cells. Using PLD1 and PLD2 siRNA, we were able to partially decrease the expression of PLD1 (by 42%) and PLD2 (by30 %). Since PLD-generated PA activates mTORC1, the main inhibitor of autophagy initiation, we wanted to study the effect of classical PLDs silencing on mTOR activation. For this purpose, WB assays were performed in order to study the mTOR downstream effector S6 kinase (S6K) activation (phosphorylation) in ABC cells transfected with PLD1 and PLD2 siRNA. Our results show that in ABC cells transfected with PLD1 and PLD2 siRNA S6K activation is reduced by 34%. This result is in accordance with the increased autophagic process induced by PLD1 and PLD2 pharmacological inhibitors, previously observed in D407 RPE cells. Since it was previously demonstrated that the PLD pathway can modulate the phagocytic process in macrophages, we wanted to evaluate the effect of PLD1 and PLD2 silencing on the photoreceptor outer segment (POS) phagocytic process in ABC cells. With this aim, ABC cells were incubated with POS for 16 h, and total (bound + internal) and internalized POS were measured by WB using an anti-rhodopsin antibody. Under basal conditions, PLD1 and PLD2 silencing seem not to significantly affect POS phagocytosis by ABC cells. In conclusion, our results demonstrate the expression of classical PLD isoforms in a new RPE cell line and their role in the modulation of the mTOR/S6K pathway. Further experiments are needed to fully elucidate the role of classical PLDs in the phagocytic process of ABC RPE cells exposed to inflammatory conditions. The results presented herein, together with our previous findings, contribute to the knowledge of the molecular basis of retinal inflammatory and degenerative diseases, such as diabetic retinopathy, aged-related macular degeneration and endophthalmitis, among others.Fil: Bermúdez, Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Asatryan, Asatryan A.. Louisiana State University Health Sciences Center New Orleans (lsu Health);Fil: Mukherjee, P. K.. Louisiana State University Health Sciences Center New Orleans (lsu Health);Fil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Bazan, Nicolás Guillermo. Louisiana State University Health Sciences Center New Orleans (lsu Health);Fil: Mateos, Melina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaLVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General MicrobiologyArgentinaSociedad Argentina de Investigación en Bioquímica y Biología MolecularSociedad Argentina de Microbiología GeneralTech Science Press2021info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/vnd.openxmlformats-officedocument.wordprocessingml.documentapplication/pdfhttp://hdl.handle.net/11336/229410Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line; LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology; Argentina; 2020; 74-750327-9545CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.techscience.com/biocell/v45nSuppl.1/42376/pdfNacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:11:17Zoai:ri.conicet.gov.ar:11336/229410instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:11:17.911CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line
title Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line
spellingShingle Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line
Bermúdez, Vicente
PHOSPHOLIPASE D
PHAGOCYTOSIS
RETINAL PIGMENT EPITHELIUM
title_short Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line
title_full Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line
title_fullStr Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line
title_full_unstemmed Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line
title_sort Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line
dc.creator.none.fl_str_mv Bermúdez, Vicente
Asatryan, Asatryan A.
Mukherjee, P. K.
Giusto, Norma Maria
Bazan, Nicolás Guillermo
Mateos, Melina Valeria
author Bermúdez, Vicente
author_facet Bermúdez, Vicente
Asatryan, Asatryan A.
Mukherjee, P. K.
Giusto, Norma Maria
Bazan, Nicolás Guillermo
Mateos, Melina Valeria
author_role author
author2 Asatryan, Asatryan A.
Mukherjee, P. K.
Giusto, Norma Maria
Bazan, Nicolás Guillermo
Mateos, Melina Valeria
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv PHOSPHOLIPASE D
PHAGOCYTOSIS
RETINAL PIGMENT EPITHELIUM
topic PHOSPHOLIPASE D
PHAGOCYTOSIS
RETINAL PIGMENT EPITHELIUM
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The retinal pigment epithelium (RPE) plays critical roles in the correct function of the neural retina and photoreceptor survival. Classical phospholipases D (PLD1 and 2) hydrolyze phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline. PA can be further dephosphorylated to diacylglycerol (DAG) by lipid phosphate phosphatases (LPPs). DAG and PA, as bioactive lipids, can modulate the activity of various proteins involved in cell signaling events, such as protein kinases C and the mTOR (mammalian target of rapamycin) complex, among others. Our previous studies demonstrated for the first time the participation of classical PLDs in the inflammatory response and in the autophagic process of RPE (ARPE-19 and D407) cells exposed to lipopolysaccharide (LPS). The aim of the present work was to study PLD1 and PLD2 expression in a new human RPE cell line (ABC cells), spontaneously arisen from a primary RPE cell culture. Western blot assays (WB) show that both classical PLDs are expressed in ABC cells. Using PLD1 and PLD2 siRNA, we were able to partially decrease the expression of PLD1 (by 42%) and PLD2 (by30 %). Since PLD-generated PA activates mTORC1, the main inhibitor of autophagy initiation, we wanted to study the effect of classical PLDs silencing on mTOR activation. For this purpose, WB assays were performed in order to study the mTOR downstream effector S6 kinase (S6K) activation (phosphorylation) in ABC cells transfected with PLD1 and PLD2 siRNA. Our results show that in ABC cells transfected with PLD1 and PLD2 siRNA S6K activation is reduced by 34%. This result is in accordance with the increased autophagic process induced by PLD1 and PLD2 pharmacological inhibitors, previously observed in D407 RPE cells. Since it was previously demonstrated that the PLD pathway can modulate the phagocytic process in macrophages, we wanted to evaluate the effect of PLD1 and PLD2 silencing on the photoreceptor outer segment (POS) phagocytic process in ABC cells. With this aim, ABC cells were incubated with POS for 16 h, and total (bound + internal) and internalized POS were measured by WB using an anti-rhodopsin antibody. Under basal conditions, PLD1 and PLD2 silencing seem not to significantly affect POS phagocytosis by ABC cells. In conclusion, our results demonstrate the expression of classical PLD isoforms in a new RPE cell line and their role in the modulation of the mTOR/S6K pathway. Further experiments are needed to fully elucidate the role of classical PLDs in the phagocytic process of ABC RPE cells exposed to inflammatory conditions. The results presented herein, together with our previous findings, contribute to the knowledge of the molecular basis of retinal inflammatory and degenerative diseases, such as diabetic retinopathy, aged-related macular degeneration and endophthalmitis, among others.
Fil: Bermúdez, Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Asatryan, Asatryan A.. Louisiana State University Health Sciences Center New Orleans (lsu Health);
Fil: Mukherjee, P. K.. Louisiana State University Health Sciences Center New Orleans (lsu Health);
Fil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
Fil: Bazan, Nicolás Guillermo. Louisiana State University Health Sciences Center New Orleans (lsu Health);
Fil: Mateos, Melina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina
LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology
Argentina
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Sociedad Argentina de Microbiología General
description The retinal pigment epithelium (RPE) plays critical roles in the correct function of the neural retina and photoreceptor survival. Classical phospholipases D (PLD1 and 2) hydrolyze phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline. PA can be further dephosphorylated to diacylglycerol (DAG) by lipid phosphate phosphatases (LPPs). DAG and PA, as bioactive lipids, can modulate the activity of various proteins involved in cell signaling events, such as protein kinases C and the mTOR (mammalian target of rapamycin) complex, among others. Our previous studies demonstrated for the first time the participation of classical PLDs in the inflammatory response and in the autophagic process of RPE (ARPE-19 and D407) cells exposed to lipopolysaccharide (LPS). The aim of the present work was to study PLD1 and PLD2 expression in a new human RPE cell line (ABC cells), spontaneously arisen from a primary RPE cell culture. Western blot assays (WB) show that both classical PLDs are expressed in ABC cells. Using PLD1 and PLD2 siRNA, we were able to partially decrease the expression of PLD1 (by 42%) and PLD2 (by30 %). Since PLD-generated PA activates mTORC1, the main inhibitor of autophagy initiation, we wanted to study the effect of classical PLDs silencing on mTOR activation. For this purpose, WB assays were performed in order to study the mTOR downstream effector S6 kinase (S6K) activation (phosphorylation) in ABC cells transfected with PLD1 and PLD2 siRNA. Our results show that in ABC cells transfected with PLD1 and PLD2 siRNA S6K activation is reduced by 34%. This result is in accordance with the increased autophagic process induced by PLD1 and PLD2 pharmacological inhibitors, previously observed in D407 RPE cells. Since it was previously demonstrated that the PLD pathway can modulate the phagocytic process in macrophages, we wanted to evaluate the effect of PLD1 and PLD2 silencing on the photoreceptor outer segment (POS) phagocytic process in ABC cells. With this aim, ABC cells were incubated with POS for 16 h, and total (bound + internal) and internalized POS were measured by WB using an anti-rhodopsin antibody. Under basal conditions, PLD1 and PLD2 silencing seem not to significantly affect POS phagocytosis by ABC cells. In conclusion, our results demonstrate the expression of classical PLD isoforms in a new RPE cell line and their role in the modulation of the mTOR/S6K pathway. Further experiments are needed to fully elucidate the role of classical PLDs in the phagocytic process of ABC RPE cells exposed to inflammatory conditions. The results presented herein, together with our previous findings, contribute to the knowledge of the molecular basis of retinal inflammatory and degenerative diseases, such as diabetic retinopathy, aged-related macular degeneration and endophthalmitis, among others.
publishDate 2021
dc.date.none.fl_str_mv 2021
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dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/229410
Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line; LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology; Argentina; 2020; 74-75
0327-9545
CONICET Digital
CONICET
url http://hdl.handle.net/11336/229410
identifier_str_mv Phospholipase D (PLD) 1 and 2 expression in ABC cells, a new retinal pigment epithelium cell line; LVI Annual Meeting Argentine Society for Biochemistry and Molecular Biology and XV Annual Meeting Argentinean Society for General Microbiology; Argentina; 2020; 74-75
0327-9545
CONICET Digital
CONICET
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